Genome-wide identification of conserved microRNA and their response to drought stress in Dongxiang wild rice (Oryza rufipogon Griff.)

Objectives

To identify drought stress-responsive conserved microRNA (miRNA) from Dongxiang wild rice (Oryza rufipogon Griff., DXWR) on a genome-wide scale, high-throughput sequencing technology was used to sequence libraries of DXWR samples, treated with and without drought stress.

Results

505 conserved miRNAs corresponding to 215 families were identified. 17 were significantly down-regulated and 16 were up-regulated under drought stress. Stem-loop qRT-PCR revealed the same expression patterns as high-throughput sequencing, suggesting the accuracy of the sequencing result was high. Potential target genes of the drought-responsive miRNA were predicted to be involved in diverse biological processes. Furthermore, 16 miRNA families were first identified to be involved in drought stress response from plants.

Conclusion

These results present a comprehensive view of the conserved miRNA and their expression patterns under drought stress for DXWR, which will provide valuable information and sequence resources for future basis studies.

     

原、异位保存东乡野生稻主要农艺性状的比较研究

为了解原、异位保存东乡野生稻主要农艺性状的变化,对庵家山普通野生稻原、异位群体进行了考察和比较分析。结果表明,原、异位保存的东乡野生稻在每穗总粒数、每穗实粒数、结实率等方面存在较大的差异,如异位保存的东乡野生稻每穗总粒数平均为58.28粒,比原位多11.44粒,但其每穗实粒数却较少,为9.89粒,结实率降低了45.43%。从种植环境可以发现,异位保存圃的环境不适宜东乡野生稻的生长,应该选择与原生境相类似的种植环境,以便于东乡野生稻的保存、研究与利用。     

东乡野生稻BILs群体耐低氮性表型性状指标筛选及其综合评价

为了鉴定东乡野生稻及其后代群体的耐低氮性,研究低氮和正常氮2种处理下“协青早B//东乡野生稻/协青早B”BC₁-F₁₂回交重组自交系株高、抽穗期、穗长、有效穗数、穗实粒数、穗总粒数、着粒密度、结实率、千粒重和单株产量等10个表型性状,利用主成分分析和模糊隶属函数对BILs群体的耐低氮性进行综合评价.结果表明： 株系116、143和157的耐低氮性强,可作为东乡野生稻耐低氮性遗传研究和水稻耐低氮性育种的中间材料.采用逐步回归分析法建立了耐低氮性最优回归方程,筛选到株高、穗总粒数、结实率、千粒重和单株产量等5个性状相对值可作为水稻全生育期耐低氮性的综合评价指标.因此,在水稻耐低氮性遗传改良中,应注重对这5个性状,尤其是穗总粒数和单株产量相对值的选择.
     

OsSIDP366, a DUF1644 gene,positively regulates responses to drought and salt stresses in rice

Domain of unknown function 1644(DUF1644) is a Rhighly conserved amino acid sequence motif present only in plants. Analysis of expression data of the family of DUF1644-containing genes indicated that they may regulate responses to abiotic stress in rice. Here we present our discovery of the role of Os SIDP366, a member of the DUF1644 gene family, in response to drought and salinity stresses in rice. Transgenic rice plants overexpressing Os SIDP366 showed enhanced drought and salinity tolerance and reduced water loss as compared to that in the control, whereas plants with downregulated Os SIDP366 expression levels using RNA interference(RNAi) were more sensitive to salinity and drought treatments. The sensitivity to abscisic acid(ABA)treatment was not changed in Os SIDP366-overexpressing plants, and Os SIDP366 expression was not affected in ABAdeficient mutants. Subcellular localization analysis revealed that Os SIDP366 is presented in the cytoplasmic foci that colocalized with protein markers for both processing bodies(PBs) and stress granules(SGs) in rice protoplasts. Digital gene expression(DGE) profile analysis indicated that stress-related genes such as SNAC1, Os HAK5 and PRs were upregulated in Os SIDP366-overexpressing plants. These results suggest that Os SIDP366 may function as a regulator of the PBs/SGs and positively regulate salt and drought resistance in rice.     

东乡野生稻苗期响应低温胁迫的转录组分析

为了解东乡野生稻（Oryza rufipogon）对低温胁迫的响应机制,对苗期的RNA-seq转录表达谱进行了研究。结果表明,与对照相比,共检测到10 200个差异表达基因（DEGs）,其中5 201个上调表达,4 999个下调表达,其中有426个DEGs位于已报道的水稻耐冷QTL区间,且37个为耐冷调控相关的家族基因。GO功能分类和KEGG代谢路径分析表明,核酸结合转录因子活性、氨基酸生物合成以及光合作用代谢等均参与响应低温胁迫过程。实时荧光定量分析表明,ABA响应蛋白基因、MYB转录因子和40S核糖体蛋白SA基因等12个可能与低温胁迫响应相关的DEGs表达模式与RNA-seq的一致。可见,植物激素传导途径和转录因子相关调控基因在东乡野生稻苗期响应低温胁迫过程中起重要作用。     

一个陆地棉bZIP蛋白cDNA的克隆及表达分析

利用PCR筛选方法从陆地棉纤维cDNA文库中筛选到一个全长cDNA序列,命名为GhbZIP。其编码产物长度为645个氨基酸残基,序列中含有两个未知功能的保守区域DUF630和DUF632,而DUF632区中有一个类似碱性亮氨酸拉链基元;此外氨基酸序列中还存在一个富脯氨酸区和一个富苯丙氨酸区,因此该蛋白具有植物碱性亮氨酸拉链蛋白的结构特征。亲水性分析表明,GhbZIP为一个典型的膜蛋白。GhbZIP基因主要是在开花3d之后在胚珠和纤维细胞中表达,这表明该基因可能与棉纤维伸长过程中的基因表达调控有关。     

东乡野生稻内生放线菌分离及菌株S123次级代谢产物分析

【目的】从东乡野生稻(Oryza rufipogon)中分离和鉴定内生放线菌,对其进行抗菌活性筛选,并分析高抗菌活性菌株S123的次级代谢产物。【方法】采用S培养基对东乡野生稻内生放线菌进行分离、纯化,并构建16S rRNA基因序列系统发育进化树进行菌株鉴定。以琼脂扩散法和菌丝生长速率法进行抗菌活性筛选,同时设计简并引物检测菌株I型聚酮合酶(PKS-I)基因。对具广谱抗菌活性的菌株S123进行分批大量发酵,运用多种色谱方法对发酵产物进行分离、纯化,利用MS和NMR分析鉴定化合物的结构。【结果】从东乡野生稻中共分离到11株内生放线菌,分别属于链霉菌属(8株)和假诺卡氏属(3株)。其中有8株具有抗菌活性,8株呈现I型PKS阳性。从高抑菌活性菌株S123中分离到化合物Nigericin和17-O-demethylgeldanamycin,其中Nigericin对金黄色葡萄球菌、枯草芽孢杆菌及水稻纹枯病菌均有抑制活性。【结论】对东乡野生稻内生放线菌进行了分离、鉴定和抗菌活性筛选,并从中分到两种与I型PKS基因相关活性的化合物Nigericin和17-O-demethylgeldanamycin,为研究东乡野生稻内生放线菌的多样性和次级代谢产物的分离提供依据。     

东乡野生稻耐冷渐渗系的细胞学观察及SSR分析

以前期鉴定筛选的2个东乡野生稻强耐冷渐渗系(IL5243和IL5335)为试材,研究其减数分裂时期的染色体行为特征及外源基因的渗入分子证据。结果表明:(1)IL5243和IL5335中正常减数分裂的花粉母细胞分别达89.93%和90.22%,最终形成正常的成熟花粉粒,花粉离体萌发率分别为(83.03±2.82)%和(81.96±1.73)%,与受体亲本无显著性差异。(2)在减数分裂I中,2个耐冷渐渗系均观察到低频率异常染色体行为,如单价体、"8"字型二价体、多价体,以及后期I有少数花粉母细胞(3.95%～5.15%)存在落后染色体等,表明其染色体组之间发生了交换和重组;在粗线期,2个强耐冷渐渗系均观察到较高频率(IL5243和IL5335分别为27.0%和38.9%)的双核仁,而其双亲都是单核仁。(3)SSR标记和Structure分析进一步证实了栽培稻和野生稻染色体组间发生了交换重组,东乡野生稻部分DNA片段已渗入到强耐冷渐渗系中,这为水稻耐冷基因的挖掘与利用奠定了重要基础。     

Overexpression of a homopeptide repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis

Dongxiang Wild Rice (Oryza rufipogon) is the northernmost wild rice in the world known to date and has extremely high cold tolerance and many other adversity-resistant properties. To identify the genes responsible for the high stress tolerance, we isolated and characterized a basic helix-loop-helix (bHLH) protein gene OrbHLH001 from Dongxiang Wild Rice. The gene encodes an ICE1-like protein containing multiple homopeptide repeats. Expression of OrbHLH001 is induced by salt stress and is predominant in the shoots of wild rice seedlings. Overexpression of OrbHLH001 enhanced the tolerance to freezing and salt stresses in transgenic Arabidopsis. Examination of the expression of cold-responsive genes in transgenic Arabidopsis showed that the function of OrbHLH001 differs from that of ICE1 and is independent of a CBF/DREB1 cold-response pathway.     

Use of intercellular washing fluid to investigate the secreted proteome of the rice–<Emphasis Type="Italic">Magnaporthe</Emphasis> interaction

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice–Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.     

人类高度进化保守基因hnulp1中转录抑制区段的定位

hnulp1是具有碱性螺旋-环-螺旋(bHLH)的新的一类转录因子.其C端含一个DUF654结构域,其序列在同源基因中相当保守,但该结构域功能未知.利用GAL4转录因子中的DNA结合结构域(DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4-Luc)质粒,构建了哺乳动物细胞转录因子活性分析系统,随后利用GAL4-Luc荧光素酶报告基因对5种含DUF654结构域的不同缺失片段转录抑制活性进行检测.检测结果表明,该基因DUF654结构域中从Δ228-407氨基酸区段具有强烈的转录抑制活性.该结果为进一步研究DUF654结构域的功能和hnulp1基因转录调控的机制奠定了基础.     

Characterization of ZmCOLD1, novel GPCR-Type G Protein genes involved in cold stress from Zea mays L. and the evolution analysis with those from other species

Maize is one of the most vital staple crops worldwide. G proteins modulate plentiful signaling pathways, and G protein-coupled receptor-type G proteins (GPCRs) are highly conserved membrane proteins in plants. However, researches on maize G proteins and GPCRs are scarce. In this study, we identified three novel GPCR-Type G Protein (GTG) genes from chromosome 10 (Chr 10) in maize, designated as ZmCOLD1-10A, ZmCOLD1-10B and ZmCOLD1-10C. Their amino acid sequences had high similarity to TaCOLD1 from wheat and OsCOLD1 from rice. They contained the basic characteristics of GTG/COLD1 proteins, including GPCR-like topology, the conserved hydrophilic loop (HL) domain, DUF3735 (domain of unknown function 3735) domain, GTPase-activating domain, and ATP/GTP-binding domain. Subcellular localization analyses of ZmCOLD1 proteins suggested that ZmCOLD1 proteins localized on plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, amino acid sequence alignment verified the conservation of the key 187th amino acid T in maize and other wild maize-relative species. Evolutionary relationship among plants GTG/COLD1 proteins family displayed strong group-specificity. Expression analysis indicated that ZmCOLD1-10A was cold-induced and inhibited by light. Together, these results suggested that ZmCOLD1 genes had potential value to improve cold tolerance and to contribute crops growth and molecular breeding.     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

Identification of a Drought Tolerant Introgression Line Derived from Dongxiang Common Wild Rice (O. rufipogon Griff.)

Construction of introgression lines using cultivated rice as recipient and wild rice is a novel approach to explore primitive and broad genetic resources in rice breeding. We recently generated a set of 159 introgression lines via a backcrossing program using an elite Indica cultivar rice Guichao 2 (O. sativa L. ssp. indica) as recipient and a common wild rice Dongxiang accession (O. rufipogon Griff.) as donor. In this study, we have evaluated the previously constructed 159 introgression lines for drought-tolerance. A total of 12 quantitative trait loci (QTLs) related to drought tolerance were mapped. Furthermore, a drought tolerant introgression line, IL23, was identified and characterized. Genotype analysis of IL23 demonstrated that IL23 contained two QTLs associated with drought tolerance, qSDT2-1 and qSDT12-2, which were located on chromosome 2 and 12 within the two introgressed segments derived from the common wild rice, respectively. Physiological characterization, including measurement of water loss, osmotic potential, electrolytical leakage, MDA content, soluble sugars content and the leaf temperature, revealed that IL23 showed the characteristics associated with drought tolerance. Identification and characterization of IL23 would provide a useful basis for isolation of novel genes associated with drought tolerance and for molecular breeding of drought tolerant rice. Furthermore, the results in this study indicated that construction of introgression lines from common wild rice should be an appropriate approach to obtain favorable genetic materials.     

Kank proteins: a new family of ankyrin-repeat domain-containing proteins

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.